Low pressure gas accelerated gene gun

ABSTRACT

The present invention relates to a gene gun and the application of the gene gun for gene transformation. A low pressure gas is used in the gene gun to directly accelerate the biological material containing solution, so that the biological materials penetrate through the cell membrane/wall or the skin of an animal, without using metal particle carriers, for gene transformation.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the priority benefits of U.S. provisionalapplication titled” “GOLD PARTICLE-FREE GENE GUN BY FLUIDFLOW-BOMBARDMENT” filed on Feb. 3. 2003, serial No. 60/444,775. Alldisclosure of this application is incorporated herein by reference.

BACKGROUNDING OF THE INVENTION

[0002] 1. Field of Invention

[0003] The present invention relates to a gene gun system. Moreparticularly, the present invention relates to a gene gun system and theapplication of the gene gun system for gene transformation, whereinunder a low pressure, the gene gun system using a gas is able toaccelerate the biological material containing solution to a high speed,so that the biological materials penetrate through the cellmembrane/wall or skin of a animal, without using metal particle carriersand gene transformation is accomplished with only low pressure.

[0004] 2. Description of Related Art

[0005] Following the discovery of the genetic materials and rapiddevelopments in genetics, scientists now can skillfully practice geneticengineering technology, for example, introducing foreign genes tocontrol the natures of a cell or even an organism. It has been widelyapplied in basic scientific studies and in improvements of agriculturalproducts that heterologous genetic materials, such as DNAs, aretransferred to the host cells in order to change the biologicalcharacteristics and morphology. For instance, genetic engineering helpsto improve the insect resistance, the frost resistance or the nutrientcompositions in agricultural products. In recent years, the genetransformation technology, in the forms of gene therapy or genevaccines, has been applied in treating human diseases. Due to thesuccessful application of the gene transformation technology in themedical field, clinical treatments of quite a few genetic diseasesincluding cancers have significant breakthroughs.

[0006] Gene transformation can be conducted in several differentapproaches and the earliest approach is to use bacterial plasmids orviral vectors as medium carriers for delivering genes into thecytoplasma of the animal or plant cells. Though constantly developed andimproved, it is difficult to overlook the side effects, includingnon-specific immune responses and genetic recombination, of thebacterial plasmids or viral vectors and the resultant risks when appliedin medical treatments. On the other hand, free of using bacterial orviral carriers, other physical or mechanical approaches of genetransformation, such as, electro-perforation and micro-injection, canavoid the side effects of these carriers and be applied for therapeuticpurposes. However, the stability and success rate are low and theoperation is laborious.

[0007] In recent years, gene transformation using a physical approachhas been applied in empirical practices. The physical approach, particlegun, is to accelerate gold particles carrying the biological materials(e.g. DNAs) into the cells for gene transformation or gene transfer.Particle gun is also applicable to the research and development of otherfields, for example, plants, mammalian somatic cells, gene therapy, andthe recently developed DNA vaccination.

[0008] The particle gun system uses DNA-coated gold particles within asample cartridge and a gas to accelerate the tube based on the highpressure shock wave principle. When a preset high pressure is reached inthe pressurized chamber, the sample cartridge having DNA-coatedparticles is accelerated by a resulting shock wave into a stoppingscreen. The DNA-coated particles continue to accelerate to enter thetarget tissue due to the inertia effect. A major disadvantage of theaforementioned methods is the loud noise resulting from the shock wave.The high speed and high pressure gas that is generated by the shock wavealso causes cell deaths. Moreover, the gas used in the conventionalparticle gun technique employs the expensive helium gas and costly goldparticles are required as carriers.

[0009] Although it is feasible to use particle gun using the genecarrying gold particles, the gold particles regularly cause damages tothe cells or the interactions between the metal particles and thecarried biological materials induce structural changes of the biologicalmaterials, thus hampering the clinical effects. Moreover, the operationof the particle gun is inconvenient to the user, because the preparationof the DNA-coated gold particles is complicated and troublesome. Fortherapeutic purposes, an effective and convenient gene delivery tool isneeded for specific attenuated vaccines expressed through the epidermaltissues, virions for gene therapy or reagents for clinical tests.Especially, this gene delivery tool can dependably deliver the geneticor biological materials without causing significant variations.

SUMMARY OF THE INVENTION

[0010] Based on the theory of aerodynamic and the bi-phase flow theory,the present invention provides a low-pressure gas accelerated gene gun,wherein the problems of low noise, cell death induced by shock wave andthe application of the expensive helium gas or gold particles areprevented. The gene gun of this invention can be applied to delivervarious kinds of biological or genetic materials into the cell.

[0011] To resolve the aforementioned problems, the present inventionprovides a gene gun, which is operable under a low pressure, using a gas(a nitrogen gas, a helium gas or even air) to directly acceleratebiological material containing solution to an extreme high speed, forexample, greater than 200 m/sec, free of using the micro-carriers (goldparticles or tungsten particles). The biological materials can penetratethrough the epidermal tissues of animal or the cell membrane/wall andenter into cytoplasma or the cell nucleus to express the special proteinand to generate the new biological function.

[0012] The present invention provides a gene gun, wherein the gene gunis applicable in gene transformation. According to a preferredembodiment of the present invention, the contour design of the spraynozzle of the gene gun and modification of the gene gun operation allowsthe gene gun to operate at a pressure lower than 4 atm. and acceleratethe sample solution to an extreme high speed. Without being carried bythe metal particles, the biological materials can penetrate theepidermis or the cell/membrane/wall and enter into the cell or theorganism.

[0013] Since a low pressure is used, the biological materials are driveninto the cell without using metal particles, to achieve genetransformation with minimal noise and damages to the cells. Moreover,due to the lower pressure for the gene gun operation, the consumption ofthe gas and the operation cost is reduced along with decreased noise, inthe present invention.

[0014] Since the sample of this invention is prepared in the solutionform without using metal particles, the operation of the gene gun iseasy and straightforward, disregarding the difficulties conventionallyencountered in the preparation of the gold particles. In the presentinvention, the biological material, for example, DNAs, RNAs, proteins,virions or drugs, is prepared in the solution form and accelerated toenter into the cell for delivery or gene transformation. Since theoperation of the gene gun free of metal particles for carrying thebiological materials, risks of altering the biological material arelowered and damages to the target cells are lessened. The gene gun ofthis invention can deliver the biological material into the cell or theorganism, and can be applied in the fields of immuno-vaccines,immuno-therapy, cancer treatments or gene therapy.

[0015] It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary, andare intended to provide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The file of this patent contains at least one drawing executed incolor. Copies of this patent with color drawing(s) will be provided bythe Patent and Trademark Office upon request and payment of thenecessary fee. The accompanying drawings are included to provide afurther understanding of the invention, and are incorporated in andconstitute a part of this specification. The drawings illustrateembodiments of the invention and, together with the description, serveto explain the principles of the invention.

[0017]FIG. 1 is a cross-sectional view of a gene gun according to apreferred embodiment of the present invention.

[0018]FIG. 2 illustrates the connection between the gene gun and tubeassembly.

[0019]FIG. 3 is a cross-section view of a sprayer and a spray nozzleaccording to a preferred embodiment of the present invention.

[0020]FIG. 4A is a magnified view (40×) of the abdominal epidermis cellsof a mouse subsequent to DNA solution bombardment using the gene gun ofthe present invention.

[0021]FIG. 4B is a magnified view (400×) of the abdominal epidermiscells of a mouse subsequent to DNA solution bombardment using the genegun of the present invention.

[0022]FIG. 5 is a magnified view (40×) of the abdominal epidermis cellsof a mouse subsequent to bombardment with chitosan-DNA solution usingthe gene gun of the present invention.

[0023]FIG. 6 is a magnified view (400×) of the frozen slice of abdominalepidermis cells of a mouse subsequent to bombardment with EGFP proteinsolution using the gene gun of the present invention.

[0024]FIG. 7 is a magnified view (40×) of abdominal epidermis cells of amouse subsequent to DNA solution bombardment using the gene gun of thepresent invention.

[0025]FIG. 8 is a magnified view (40×) of abdominal epidermis cells of amouse subsequent to bombardment with DNA-coated particles using the genegun of the present invention.

[0026]FIG. 9 shows the western blotting result for monitoring the immuneresponse of the mouse subsequent to DNA solution bombardment using thegene gun of the present invention.

[0027]FIG. 10 shows the western blotting result for monitoring theimmune response of the mouse subsequent to bombardment with DNA-coatedparticles using the gene gun of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Referring to FIG. 1, FIG. 1 is a cross-sectional view of a genegun 100 according to a preferred embodiment of the present invention.The gene gun 100 of the present invention is divided into four parts,comprising at least a pressurized chamber 102, a sprayer 104, a backsideconnector 106 and a material delivery system 108. The backside connector106 is connected to the high pressure gas source (FIG. 2) via valves. Agas (flow direction shown as arrow) is delivered from the backsideconnector 106 to the pressurized chamber 102. As the gas in thepressurized chamber 102 is built up to a preset pressure, thehigh-pressure gas and the sample solution carried by the high-pressuregas are sprayed out through the sprayer 104. The material of thepressurized chamber is a light metal, for example, aluminum alloy. Thematerial of the sprayer 104 is a biocompatible metal, for example,stainless steel or its alloys.

[0029] In general, the sample solution (i.e. biological materialcontaining solution) is accelerated by a gas to a velocity of about 200to 300 meter/sec, and this speed does not exceed the speed of sound. Thesprayer 104, as shown in FIG. 1, includes at least a terminal spray tube104 a and a nozzle 104 b. According to the theory of aerodynamics, asthe pressure difference between the internal and the external of thespray nozzle is greater than 1.8 atm, a supersonic flow is generated. Ifthe cross-sectional diameter of the nozzle converges initially and thendiverges to a small degree, a supersonic flow is generated in the nozzle104 b. The gas velocity gradually decreases as the gas enters the spraytube 104 a. Increasing the length of the spray tube 104 a, the gasvelocity decreases. The gas velocity can thereby be controlled within acertain limits according to the length of the spray tube 104 a.

[0030] According to a preferred embodiment of the present invention, anappropriate design includes an application of sample solution withoutmicro-carriers and a material delivery system as disclosed in thepresent invention. The sample solution can uniformly be accelerated to arequired velocity without the application of high pressure, thusmitigating the damage to the target cells. Moreover, the contour designof the spray nozzle 104 b and the spray tube 104 a allows the pressureat the exit of the spray nozzle to approach the atmospheric pressure.Damages to the target cells are thereby reduced. According to onepreferred embodiment of the present invention, the gene gun operationuses the sample solution without containing particle carriers in thesolution, in combination of a special spray nozzle of the gene gun. Inaccordance of this special spray nozzle, the sample solution can beaccelerated to different speeds by varying the length of the terminalspray tube and the type of gas source.

[0031] The design of the material delivery system 108 can be adjustedaccording to the operation requirements. As exemplified, a gravitydispensing mechanism is employed to release droplets of the samplesolution from the material delivery system. The sample solution dropletsare then carried away by the high velocity gas flow and are dischargedat a high speed into the target tissue. The sample solution can beinjected into an inner cavity of the material delivery system 108through an inlet, while the inner cavity is big enough to hold thesample solution. The sample solution flows through the channel that isconnected to the inner cavity to the outlet and is steadily released asdroplets around the spray neck. Alternatively, the material deliverysystem can further includes a delayed release loading station, so thatthe sample solution droplets are placed on the loading station, waitingto be carried away by the gas flow. Other mechanisms, such as viscositycontrol, can be used to achieve the delayed release function. Inaddition, a control valve can be incorporated into the material deliverysystem to control the release of the sample solution, or the releaseamount, depending on whether the gene gun discharges one single shot ormultiple shots.

[0032] Alternatively, the material delivery system 108 can be designedto facilitate the operation in single shot. Before shooting, the sprayneck can be separated from the pressurized chamber and the samplesolution is loaded on the spray neck. Once the spray neck is connectedwith the pressurized chamber, the sample solution is ready to bedischarged. During operation, the sample solution is exposed to thehigh-velocity gas flow. The high-velocity gas flow would carry thesample solution out of the spray neck and the sample solution is furtheraccelerated to the required velocity by the sprayer 104.

[0033]FIG. 2 illustrates the entire gene gun system. The gas source forthe gene gun system 200 is provided from a gas tank 220. The gas sourcefrom the gas tank 220 includes a helium gas, a nitrogen gas or othertype of gas or air. The helium gas can travel at velocity greater than1000 m/s. Moreover, less damage is inflicted upon the target cellbecause the mass of helium is lower. Helium is thus an ideal gas, exceptfor being too expensive. Using helium gas is essential on animal orplant cells with a thicker keratin or wax layer. However, using anitrogen gas for the gene gun is sufficient for the easily penetratedbiological system. The gas in the gas tank 220 is set at a certainpressure by a pressure regulator 222. The pressure in the pressurizedtank of the gene gun has to be greater than 1.8 atm. Due to the drop ofthe gas pressure in the tubing, the pressure regulator 222 has to set ata higher pressure.

[0034] As shown in FIG. 2, the gas is passed from the gas tank 220through the pressure regulator 222 and the tubing 210. A control valve224 is further used to determine whether the gas would enter thepressurized chamber 202 through the back-side connector 206. The controlvalve 224 is controlled by a controller (not shown). When the gene gunsystem 200 is operating, the pressure regulator 222 is set at anappropriate pressure. The biological material containing solution(sample solution) is placed in the gene gun. The biological material canbe RNAs, DNAs, proteins, peptides, saccharides, virions, or drugs, forexample. The trigger device 226 that is connected to the handlestructure 230 of the gene gun system is initiated, the controller is setoff, sending a signal to open the control valve 224. The gas flows intothe pressurized chamber 202, and after the establishment of the fluidflow field, the sample solution is then carried out by the high-velocitygas flow and is discharged into the target tissue through the sprayer204.

[0035] Though the structure of the gene gun of the present inventionincludes a handle structure and a triggering device, the handlestructure and the triggering device are not limited to any single typeof technology or assembly. Any existing assembly or technology for thehandle structure or the triggering device can be used. Since the handlestructure and the triggering device are not the essential features ofthe present invention, details for the handle structure and thetriggering device will not be reiterated. It is intended that thespecification and examples to be considered as exemplary only.Additional advantages and modifications are readily occurred to thoseskilled in the art from the consideration of the specification and thepractice of the invention disclosed herein.

[0036] In order for the sample solution droplets to travel at a highspeed, the present invention provides a high-speed sprayer 300, as shownin FIG. 3. Unlike the conventional straight spray tube, the spray tube302 of the present invention is conical shape, allowing the speed of thegas flow to achieve the supersonic flow rate and the speed of the samplesolution to approach the speed of sound. The spray nozzle 304 comprisesa contour entrance, allowing the discharged sample solution to be moreevenly distributed and are not localized at the exit, which causes celldeath. Moreover, the gas pressure at the exit of the spray nozzleapproaches atmospheric pressure to mitigate damages to the cell.

[0037] The spray tube and the spray nozzle of the present invention aredesigned according to the following theory:

[0038] Assuming the flow field is an isentropic flow, the ratio of thearea of the spray nozzle (Ae) and the area of the spray neck A* is$\frac{Ae}{A^{*}} = {\frac{1}{Me}\left\lfloor {\frac{2}{\gamma + 1}\left( {1 + {\frac{\gamma - 1}{2}{Me}^{2}}} \right)} \right\rfloor^{\frac{\gamma + 1}{2{({\gamma - 1})}}}}$

[0039] wherein, Me is the Mach number, which is a ratio of the gas flowrate over the speed of sound and γ is the specific heat ratio. If Me, Aeand γ are defined, A* is determined.

[0040] Similarly, if the pressure at the exit of the spray nozzle, thepressure in the pressurized chamber Po can also be determined accordingto the following equation${Po} = {{P\left( {1 + {\frac{\gamma - 1}{2}{Me}^{2}}} \right)}^{\frac{\gamma}{\gamma - 1}}.}$

[0041] If the pressure in the pressurized chamber is the gas tankpressure, the required pressure in the gas tank Pc is determinedaccording to the following equation${Pc} = {{P\left( {1 + {\frac{\gamma - 1}{2}{M_{in}}^{2}}} \right)}^{\frac{\gamma}{\gamma - 1}}\frac{1}{Ld}}$

[0042] wherein Ld is dump loss.

[0043] Since a certain gas flow rate is needed to carry out the samplesolution, the necessary gas tank pressure is resulted by defining theMack number at the entrance of the pressurized chamber (M_(in)).

[0044] Moreover, under the steady state condition, the mass flow rate inm_(in) is equal to the mass flow rate out m_(out),${\overset{.}{m}}_{in} = {\left( {\rho \quad {AV}} \right)_{in} = \left( {\rho \quad {AV}} \right)_{out}}$${A_{in}\frac{P_{c}}{\sqrt{T_{o}}}\sqrt{\frac{\gamma}{R}}\frac{M_{in}}{\left( {1 + {\frac{\gamma - 1}{2}M_{in}}} \right)^{\frac{\gamma + 1}{2{({\gamma - 1})}}}}} = {A^{*}\frac{P_{0}}{\sqrt{T_{0}}}\sqrt{\frac{\gamma}{R}}\sqrt{\left( \frac{2}{\gamma + 1} \right)^{\frac{\gamma + 1}{\gamma - 1}}}}$

[0045] wherein ρ is the gas density, T₀ is the temperature in thepressurized chamber, A_(in), which is area of the entrance of thepressurized chamber from the gas tank. A_(in) is thereby easilydetermined.

[0046]FIG. 3 illustrates the contour design of the spray nozzle 304 ofthe present invention. The spray nozzle 304 is designed to comprise aconverging part 32 and a diverging part 36. The transition regionbetween the converging part 32 and the diverging part 36 is the sprayneck 34. The contour of the spray nozzle 304 is obviated from any abrupttransition to allow a smooth gas flow. A general rule for designing thespray nozzle 304 of the present invention is as follow:

[0047] (1) the simplified design, wherein the gas flow rate exiting thespray nozzle is not uniform.

[0048] As shown above, R_(t) represents the curvature radius of theconverging part 32 and r_(t) is the radius of the spray neck 34, whereinr_(t)<R_(t)<2r_(t) for the converging part of the simplified design. θ,as shown above, is the angle between the diverging part 36 and centeraxis of the spray nozzle and the spray tube (broken lines), wherein θ isless than 15 degrees for the converging part of the simplified design.The contour of the converging part of the spray nozzle, is a divergingstraight tube, forming a coned shape structure. The angle θ between theslanted straight line and the center axis is less than 15 degrees, andis preferably between 10 to 15 degrees.

[0049] If the friction loss is ignored and the pressure at theconverging part is high enough, the gas flow rate should achievesupersonic in the spray tube. Due to the diverging angle θ of the spraytube, the slant shock wave caused by the supersonic gas flow can beavoided. In general, of the contour of the spray tube is fixed, theexpansion ratio of the gas flow and the Me (Mach number at the outlet)are determined. For the same spray tube, if a different gas is used, adifferent Mach number or a different pressure (P) at the inlet of thespray tube is obtained. Taking the nitrogen gas as an example, theexpansion ratio Ae/At (area of the diverging terminal/area of the neck),the relative diameter ratio De/Dt, the minimum length of the divergingpart L (1.2 degree in a corn shape) and the pressure at the inlet of thespray tube are listed in the followings.

[0050] (A) if M_(e)=1.2 , D_(e)=5 mm

[0051] P=2.425(atm.) A_(e)/A_(t)=1.034 D_(e)/D_(t)=1.0151

[0052] D_(t)=4.925 mm L=3.57 mm

[0053] (B) if M_(e)=1.3, D_(e)=5 mm

[0054] P=2.771(atm.) A_(e)/A_(t)=1.0663 D_(e)/D_(t)=1.0326

[0055] D_(t)=4.842 mm L=7.58 mm

[0056] (C) if M_(e)=1.4, D_(e)=5 mm

[0057] P=3.182(atm.) A_(e)/A_(t)=1.1115 D_(e)/D_(t)=1.0543

[0058] D_(t)=4.7425 mm L=12.356 mm

[0059] (D) if M_(e)=1.5, D_(e)=5 mm

[0060] P=3.67(atm.) A_(e)/A_(t)=1.1762 D_(e)/D_(t)=1.0845

[0061] D_(t)=4.6104 mm L=18.7 mm

[0062] (E) if M_(e)=1.6, D_(e)=5 mm

[0063] P=4.25(atm.) A_(e)/A_(t)=1.2502 D_(e)/D_(t)=1.118

[0064] D_(t)=4.4723 mm L=25.33 mm

[0065] (F) if M_(e)=1.2, D_(e)=4 mm

[0066] P=2.425(atm.) A_(e)/A_(t)=1.034 D_(e)/D_(t)=1.0151

[0067] D_(t)=3.9405 mm L=2.856 mm

[0068] (G) if M_(e)=1.3, D_(e)=4 mm

[0069] P=2.771(atm.) A_(e)/A_(t)=1.0663 D_(e)/D_(t)=1.0326

[0070] D_(t)=3.8736 mm L=6.06 mm

[0071] (H) if M_(e)=1.4, D_(e)=4 mm

[0072] P=3.182(atm.) A_(e)/A_(t)=1.1115 D_(e)/D_(t)=1.0543

[0073] D_(t)=3.794 mm L=9.8886 mm

[0074] (I) if M_(e)=1.5, D_(e)=4 mm

[0075] P=3.67(atm.) A_(e)/A_(t)=1.1762 D_(e)/D_(t)=1.0845

[0076] D_(t)=3.688 mm L=14.96 mm

[0077] (J) if M_(e)=1.6, D_(e)=4 mm

[0078] P=4.25(atm.) A_(e)/A_(t)=1.2502 D_(e)/D_(t)=1.118

[0079] D_(t)=3.5778 mm L=20.26 mm

[0080] (K) if M_(e)=1.2, D_(e)=3 mm

[0081] P=2.425(atm.) A_(e)/A_(t)=1.034 D_(e)/D_(t)=1.0151

[0082] D_(t)=2.9554 mm L=2.14 mm

[0083] (L) if M_(e)=1.3, D_(e)=3 mm

[0084] P=2.771(atm.) A_(e)/A_(t)=1.0663 D_(e)/D_(t)=1.0326

[0085] D_(t)=2.905 mm L=4.549 mm

[0086] (M) if M_(e)=1.4, D_(e)=3 mm

[0087] P=3.182(atm.) A_(e)/A_(t)=1.1115 D_(e)/D_(t)=1.0543

[0088] D_(t)=2.845 mm L=7.416 mm

[0089] (N) if M_(e)=1.5, D_(e)=3 mm

[0090] P=3.67(atm.) A_(e)/A_(t)=1.1762 D_(e)/D_(t)=1.0845

[0091] D_(t)=2.766 mm L=11.22 mm

[0092] (O) if M_(e)=1.6, D_(e)=3 mm

[0093] P=4.25(atm.) A_(e)/A_(t)=1.2502 D_(e)/D_(t)=1.118

[0094] D_(t)=2.6834 mm L=15.198 mm

[0095] (P) if M_(e)=1.2, D_(e)=2.5 mm

[0096] P=2.425(atm.) A_(e)/A_(t)=1.034 D_(e)/D_(t)=1.0151

[0097] D_(t)=2.463 mm L=1.776 mm

[0098] (Q) if M_(e)=1.3, D_(e)=2.5 mm

[0099] P=2.771(atm.) A_(e)/A_(t)=1.0663 D_(e)/D_(t)=1.0326

[0100] D_(t)=2.426 mm L=3.565 mm

[0101] (R) if M_(e)=1.4, D_(e)=2.5 mm

[0102] P=3.182(atm.) A_(e)/A_(t)=1.1115 D_(e)/D_(t)=1.0543

[0103] D_(t)=2.371 mm L=6.180 mm

[0104] (S) if M_(e)=1.5, D_(e)=2.5 mm

[0105] P=3.67(atm.) A_(e)/A_(t)=1.1762 D_(e)/D_(t)=1.0845

[0106] D_(t)=2.305 mm L=9.350 mm

[0107] (T) if M_(e)=1.6, D_(e)=2.5 mm

[0108] P=4.25(atm.) A_(e)/A_(t)=1.2502 D_(e)/D_(t)=1.118

[0109] D_(t)=2.236 mm L=12.665 mm

[0110] From the above calculations, the pressure P, the diameter of thespray neck Dt and the minimum length of the diverging part L areobtained when the Mach number at the outlet Me and the diameter of theoutlet of the spray tube De are fixed. If Dt is fixed as 4.74 mm,pressure P needs to be 3.18 atm. (about 46.75 psi) to obtain Me=1.4.Considering the pressure loss in the whole system, the pressure of thegas after the control valve should be around 90-100 psi, still under thelow pressure range of the gas tank.

[0111] (2) the uniform gas flow rate design.

[0112] The direction of the gas flow, at the spray nozzle, is notparallel to the center axis. In order to provide a gas flow thatparallels to the center axis, the expansion wave generated in the sprayneck of the above simple design of the diverging part must becompensated by a special curvature design of the spray nozzle.

[0113] Since the gas flow rate achieves supersonic at the exit of thespray nozzle, a coned shape spray tube or a straight spray tube isconnected to the spray nozzle to accelerate the sample to high a speed,which is proven to be effective in penetrating into the epidermis cell,through the cell wall and/or the cell membrane.

[0114] Sample solution preparation. The preparation of the samplesolution can be revised and modified based on the containing biologicalmaterials, but not limited to any specific technique or procedure. Ingeneral, the biological material can be processed and then dissolved inthe suitable solution as the sample solution, without using metalparticle carriers.

[0115] Preparation of DNA Solution

[0116] DNA plasmid constructed with the complete protein expressionsystem is dissolved in the sterile distilled water. The preparedsolution can be applied directly for the bombardment of the gene gun.

[0117] Preparation of Chitosan Oligomer

[0118] Chitosan(Sigma C-3646) is dissolved in 6% acetic acid solution ina concentration of 5 mg/mL, followed by adding 8% KNO₂ (w/w ofchitosan), and reacted under room temperature for 1 hour. After 1 hour,add 0.1N NaOH in a same volume to terminate the reaction, followed byadding absolute alcohol (solution volume/absolute alcoholvolume={fraction (1/3)}), then replaced under 4° C. overnight. Themixture is centrifuged (1500 rpm) under 4° C. to collect theprecipitate, and the precipitate is re-dissolved in 0.5% acetic acidsolution in a concentration of 2 mg/mL and filtered by a 0.45 μm filter.

[0119] Preparation of Chitosan Oligomer-CMC Complex

[0120] Chitosan Oligomer is mixed with 2 mg/mL carboxymethylcellulose(CMC) in a 3:1 ratio (in volume), and vortexed to obtain the ChitosanOligomer-CMC complex.

[0121] Preparation of DNA-chitosan Oligomer-CMC Complex

[0122] 0.3 μl (concentration 1 μg/μl ) pEGFP-N2 vector (Clontech) isadded with 4 μl Chitosan Oligomer-CMC complex and the mixture isvortexed until homogenous.

[0123] Bombardment of mouse skin with DNA solution. The specimen usedfor the gene gun experiment is BALB/c mice of about a few weeks old.Hair at the abdomen of the mouse is shaved to expose the skin of themouse. 3-4 week-old Balb/c mice are selected and shaved to expose theabdominal skin. After filling naked DNA or chitosan-CMC-DNA solutioninto the gene gun, bombardment is performed to the exposed mice skin.The size of the abdominal skin for each mouse can take average fourshots of bombardment, with each shot in a volume of 4.3 μl. After oneday, the mice are sacrificed to remove the skin for observation underfluorescent microscopy or under microtomy.

[0124] Bombardment of mouse skin with EGFP protein. Similar to thebombardment procedure with DNA solution, mice are shaved to expose theabdominal skin. After filling EGFP protein (4 μg) solution into the genegun, bombardment is performed to the exposed mice skin. After thebombardment, the skin is washed with cleaning solution to wash off theprotein remained outside the skin. Afterwards, the mice are sacrificedto remove the skin for observation under fluorescent microscopy or undermicrotomy.

[0125] Frozen section of mice skin. The mice skin is removed andsectioned into cubes, treated with O.C.T. embedding agent (Tissue-Tek,Sakura Finetechnical Co. Ltd.). The tissue is then sectioned into slicesof 10 μm thickness by deep-frozen microtome, observed under fluorescentmicroscopy.

[0126] Genetic immunization of Naked DNA bombardment. 3 week-old Balb/cmice are selected and the abdominal skin of the mice is bombarded by thegene gun with 24 μl DNA solution (containing 1.8 μg naked pEGFP DNAvector) once per week for 4 weeks. The bombardment is performed with ahelium pressure of 100 psi. From the 1st to 5th week, blood serum iscollected by withdrawing blood from the eye socket once per week. Serumis analyzed by Western Blotting. EGFP protein (0.5 μg) is separated byelectrophoresis (SDS-PAGE), transferred to the nitrocellulose paper anddiluted 100 times by using the serum obtained from the mice, washedthree times with TBS buffer (Tris-base 20 mM, NaCl 150 mM, Tween 200.3%, pH 7.2) and then treated with anti-mouse IgG conjugated peroxidase(Sigma A-5906). Next, the product is washed four times by TBS buffer andthe nitrocellulose paper is developed with LumiGLO™ (KPL 50-60-00 and50-59-60) for display and recordation.

[0127] Results from the Bombardment of the Gene Gun

[0128] Bombardment with Naked DNA Solution

[0129] Shooting (bombardment) is performed with a helium pressure of 60psi, while each shot includes 0.2 μg DNA. The green fluorescence presenton the abdominal epidermis cells of the mice is observed underfluorescence microscopy. As shown in FIG. 4A (40 times magnified, 40×),the left part is the shot region, emitting green fluorescence under thefluorescent microscopy; while the right part is the non-shooting region(not being shot), thus showing no fluorescence. For the shot region ofthe abdominal region, as shown in FIG. 4B (400×), almost all theepidermis cells show green fluorescence, indicating high efficacy forthe bombardment of the gene gun.

[0130] Bombardment with chitosan-DNA Nanoparticles

[0131] Shooting is performed with a helium pressure of 50 psi, whileeach shot includes 0.3 μg DNA (chitosan: CMC=3:1). The greenfluorescence present on the abdominal epidermis cells of the mice isobserved under fluorescence microscopy. The fluorescent region in FIG. 5(40×) shows the result of the bombardment with DNA-chitosannanoparticles. The distribution of green fluorescence is extensive fromthe bombardment of the gene gun, indicating a desired result of thebombardment.

[0132] Bombardment with EGFP Protein

[0133] Shooting is performed with a helium pressure of 100 psi, whileeach shot includes 4 μg EGFP protein. The fluorescence present on theabdominal skin of the mice is observed by fluorescent microscopy. Fromthe observed fluorescence result of FIG. 6 (400×) for the frozen sectionof the mice skin, it indicates that the EGFP protein is introduced intothe epidermis, but not into the deeper tissues (e.g. muscle tissue).

[0134] Gene Transformation with Naked DNA Solution or DNA CoatedParticles

[0135] 4 week-old Balb/c mice are selected and the abdominal skin of themice is bombarded by the gene gun with a helium pressure of 60 psi,using either the naked DNA sample solution without metal particlecarriers (DNA solution group) or DNA-coated gold particles sample(particle group). The particle-free DNA solution contains 0.2 μgpEGFP-N2 vector, while the DNA-coated particle sample includes 0.2 μgpEGFP-N2 vector/0.1 mg gold particles coated by spermidine/CaCl₂. Thenext day after the bombardment, the abdominal skin of the mice from twogroups is removed and observed the fluorescence under fluorescencemicroscopy.

[0136]FIG. 7 is a magnified view (40×) of abdominal epidermis cells of amouse subsequent to DNA solution bombardment using the gene gun of thepresent invention. The fluorescence present in the cells representssuccessful transformation by the pEGFP-N2 vector for the cells. Theobserved fluorescence is prominent and uniform.

[0137]FIG. 8 is a magnified view (40×) of abdominal epidermis cells of amouse subsequent to bombardment with DNA-coated particles. On the otherhand, the observed fluorescence is weak and distributes sparsely.

[0138] Compare the results of the above two groups, it shows that abetter transformation efficacy is achieved by the bombardment of thegene gun using the naked DNA solution.

[0139] Western Blotting of Mice Genetic immunization by Naked DNA

[0140] 4 week-old Balb/c mice are selected and shaved to expose theabdominal skin. After filling naked DNA sample solution (solution group)or DNA-coated gold particles (particle group) into the gene gun,bombardment is performed to the mice skin and the resultant immuneresponses from two groups are compared.

[0141] The abdominal skin of the mice is bombarded by the gene gun onceper week for 4-6 weeks and the bombardment is performed with a heliumpressure of 80 psi. For the solution group, the bombardment is performedwith naked DNA solution once per week for 4 weeks, one shot containing1.8 μg naked pEGFP-N2 vector. For the particle group, the bombardment isperformed with DNA coated gold particles once per week for 6 weeks, oneshot containing 0.6 μg naked pEGFP-N2 vector/0.6 mg gold particles. Fromthe 1st to 5th week, blood serum is collected once per week. Serum isthen analyzed by Western Blotting to see whether the anti-EGFP antibodyexists.

[0142] Results of the solution group are shown in FIG. 9. Mice m1-m5were immunized with naked DNA solution (pEGFP-N2), while mouse m6 is thecontrol, immunized with water, as having no immunization. Mice m1-m5were observed with medium antibody reaction after the second boost (the3^(rd) week), while mouse m5 was observed with weak antibody reaction inthe 2^(nd) week.

[0143] Results of the particle group are shown in FIG. 10. Mice A5 andA8 are the control, immunized with water, as having no immunization. Formice A1-A4 and A6-A7, medium antibody reaction is generally observed inthe 4^(th) or 5^(th) week, although mouse A7 is observed with smallantibody reaction in the 2^(nd) week.

[0144] In general, the immunization induced by the naked DNA solutionseems to be slightly superior, when the immunization results of the genegun with naked DNA solution and with DNA-coated particles. Both caninduce antibody reaction in 3^(rd) to 4^(th) week after the bombardment.

[0145] Accordingly, the contour design of the spray nozzle of the genegun and modification of the gene gun operation allows the gene gun tooperate at a lower pressure and accelerate the sample solution to anextreme high speed. Without being carried by the metal particles, thebiological materials can penetrate the epidermis or thecell/membrane/wall and enter into the cell or the organism.

[0146] Since a low pressure is used, the biological materials are driveninto the cell without using metal particles, to achieve genetransformation with minimal noise and damages to the cells. Moreover,since the sample of this invention is prepared in the solution formwithout using metal particles, the operation of the gene gun is easy andstraightforward, disregarding the difficulties conventionallyencountered in the preparation of the gold particles. In the presentinvention, the biological material, for example, DNAs, RNAs, proteins,virions or drugs, is prepared in the solution form and accelerated toenter into the cell for delivery or gene transformation.

[0147] Moreover, due to the contour design of the spray nozzle, theoperation of the gene gun is modified to allow an even distribution ofthe sample solution. Since the pressure at the nozzle opening is closeto atmospheric pressure, the target cell is prevented from beingdamaged.

[0148] It will be apparent to those skilled in the art that variousmodifications and variations can be made to the structure of the presentinvention without departing from the scope or spirit of the invention.In view of the foregoing, it is intended that the present inventioncover modifications and variations of this invention provided they fallwithin the scope of the following claims and their equivalents.

What is claimed is:
 1. A method for delivering a biological materialusing a gene gun, comprising: providing the gene gun comprising apressurized chamber, a sprayer, a controller and a material deliverysystem; placing a sample solution into the material delivery system,wherein the sample solution comprises at least the biological material;triggering the gene gun and providing a gas through the control of thecontroller to the pressurized chamber until the gas establishes apressure; releasing the sample solution from the material deliverysystem, so that the sample solution is accelerated by the gas in thepressurized chamber; and discharging the sample solution out of thesprayer, wherein the sprayer includes a spray nozzle and a spray tube,and the spray nozzle comprises an interior contour, wherein the interiorcontour of the spray nozzle comprises a diverging part and a convergingpart and the spray tube is a diverging straight tube, so that adischarge speed of the sample solution is supersonic and the biologicalmaterial is evenly injected into a target.
 2. The method of claim 1,wherein the biological material is a nucleic acid.
 3. The method ofclaim 1, wherein the biological material is a protein.
 4. The method ofclaim 1, wherein the biological material is a virion.
 5. The method ofclaim 1, wherein the biological material is a vaccine.
 6. The method ofclaim 1, wherein the biological material is an immunogen for cancerimmunotherapy.
 7. The method of claim 1, wherein the sample solution isaccelerated to a speed of 200-300 m/s by the gas.
 8. The method of claim1, wherein a pressure at an outlet of the sprayer is about 1 atmosphericpressure.
 9. The method of claim 1, wherein the spray nozzle furthercomprises a spray neck positioned between the diverging part and theconverging part, and a range of the interior contour of the convergingpart includes: r_(t)<R_(t)<2r_(t), wherein R_(t) represents a curvatureradius of the converging part, r_(t) is a radius of the spray neck; andwherein θ<15 degrees, wherein θ is an angle between the diverging partand a center axis of the spray tube.
 10. The method of claim 1, whereinthe gas includes a nitrogen gas or a helium gas.
 11. A method for genetransformation by using a gene gun, comprising: providing the gene guncomprising a pressurized chamber, a sprayer, a controller and a materialdelivery system; placing a sample solution into the material deliverysystem, wherein the sample solution comprises at least a nucleic acid;triggering the gene gun and providing a gas through the control of thecontroller to the pressurized chamber, wherein the gas is a nitrogen gasor a helium gas; releasing the sample solution from the materialdelivery system after the gas in the pressurized chamber establishes apressure, so that the sample solution is accelerated by the gas in thepressurized chamber; and discharging the sample solution out of thesprayer, wherein the sprayer includes a spray nozzle and a spray tube,and the spray nozzle comprises an interior contour, wherein the interiorcontour of the spray nozzle comprises a diverging part and a convergingpart and the spray tube is a diverging straight tube, so that adischarge speed of the sample solution is supersonic and the biologicalmaterial is evenly injected into a target.
 12. The method of claim 11,wherein the sample solution is accelerated to a speed of 200-300 m/s bythe gas.
 13. The method of claim 11, wherein a pressure at an outlet ofthe sprayer is about 1 atmospheric pressure.
 14. The method of claim 11,wherein the spray nozzle further comprises a spray neck positionedbetween the diverging part and the converging part, and a range of theinterior contour of the converging part includes: r_(t)<R_(t)<2r_(t),wherein R_(t) represents a curvature radius of the converging part,r_(t) is a radius of the spray neck; and wherein θ<15 degrees, wherein θis an angle between the diverging part and a center axis of the spraytube.
 15. The method of claim 1, wherein the nucleic acid is used forgene therapy.